Fermentative production of l-valine

ABSTRACT

Mutant strains of the genera Brevibacterium and Corynebacterium having a resistance to 2-thiazolalanine or having said resistance together with a nutrient requirement, produce L-valine in a culture medium.

United States Patent Tsuchida et al.

[4 1 July 8,1975

FERMENTATIVE PRODUCTION OF L-VALINE Inventors: Takayasu Tsuchida,Kawasaki;

Fumihiro Yoshinaga, Fujisawa, both of Japan Assignee: Ajinomoto Co.,Inc.,'Tokyo, Japan Filed: Mar. 1, 1974 Appl. No.: 447,379

Foreign Application Priority Data [58] Field of Search 195/28, 29, 30,47

References Cited UNITED STATES PATENTS 9/1974 Kubota et al. 195/29Primary Examiner-A. Louis Monacell Assistant Examiner-Robert J. WardenAttorney, Agent, or FirmHans Berman; Kurt Kelman [5 7] ABSTRACT Mutantstrains of the genera Brevibacterium and Corynebacterium having aresistance to 2-thiazolalanine or having said resistance together with anutrient requirement, produce L-valine in a culture medium.

SCIaims, N0 Drawings FERMENTATIVE PRODUCTION OF L-VALINE The presentinvention relates to a method of producing L-valine by bacterialfermentation.

An' object of the present invention is to produce L- valine at a lowcost from readily available raw materials by using new type ofmicroorganism. L-Valine in one of the essential amino acids for humanand animal nutrition, and a wide use thereof is'expected for example,enrichment of food, preparation of a seasoning and medical use.Proposals have been made for producing L-valine by a fermentative methodemploying a certain kind of nutrient requiring mutant strain. However,it was impossible to produce a large amount of L-valine by employingsuch a nutrient requiring mutant strain.

We have now found that some bacteria having a resistance toZ-thiazolalanine (hereinafter abbreviated as 2-TA) produce a largeamount of L-valine when cultured in a nutrient medium. We have alsofound that a mutant strain havinga resistance to 2-TA together with acertain nutrient requirement has a better ability to produce L-valinethan a strain having only resistance to 2-TA or having only a certainnutrient requirement.

A microorganism employed in the present method is a strain belonging togenera Brevibacterium and Corynebacterium which resists growthinhibition by 2-TA. The microorganism may also have anutrientrequirement and/or resistance to other reagents in addition tothe resistance to 2-TA. The required nutrient may be leucine,isoleucine, threonine, or isoleucine and methionineand the otherreagents a-amino-B-hydroxy valeric acid and ethionine.

Representative strains useful for the present invention are as follows:

1. 2-TA resistant mutants such as Corynebacterium glutamicum FERM P-1768whose parent strain is Corynebacterium glutamicum (Micrococcusglutamicus) ATCC13032, Corynebacterium acetoacidophilum FERM P-l 314whose parent strain is Corynebacterium acetoacidophilum ATCC 13870 andBrevibacterium lactofermentum FERMP-l945 whose parent strain isBrevibacterium lactofermentum ATCC 13869.

2. Mutants having nutrient requirement in addition to 2-TA resistancesuch as Corynebacterium glutamicum FERMP-1967 (leucine requiringmutant), FERMP- 1968 (threonine requiring mutant) and FERMP-1966(isoleucine requiring mutant) which were obtained by a conventionalartificial mutant inducing method from Corynebacterium glutanicumFERMP-1768 (2-TA resistant mutant); Brevibacterium lactofermentumFERMP-1964 (leucine requiring mutant), FERMP- 1963 (isoleucine requiringmutant) and FERMP-l965 (threonine requiring mutant) which were obtainedby a conventional artificial mutant inducing method from Brevibacteriumlactofermentum FERMP-1945 (Z-TA resistant mutant), and Brevibacteriumlactofermentum FERMP-1845 (isoleucine plus methionine requiring mutanthaving resistance to Z-TA) which were induced from Brevibacteriumlactofermentum FERMP-l858 (isoleucine plus methionine requiring mutant);

These mutant strains have the same morphological characteristics astheir parent strains respectively. The morphological characteristics ofCorynebacterium glutamicum ATCC 13032 are described in U.K. Patent No.839,597, and those acetoacidophilum 13870 and Brevibacteriumlactofermentum ATCC 13869 are described in US. Pat. No. 3.1 17,915.Specimen cultures of microorganisms idenof Corynebacterium tified byFERMP-number are freely available to qualifled persons from theFermentation Research Institute, Agency of Industrial Science ofTechnology, the Ministry and Industrial Trade and Industry, at l-8-5,Inage Higashi, Chiba-shi, Chiba, Japan.

The method of obtaining mutant strains employed in the present inventionis a conventional artificial mutant inducing method, and is illustrated,for example, by the following Experiment 1. The growth inhibition effectof Z-TA on a mutant having resistance to Z-TA and on its parent strain,was also examined as shown in the following Experiment 2.

EXPERIMENT 1 A culture medium containing 1 yeast extract, 1 pepton, 0.5NaCl and 0.5 glucose, of pH 7.0 was prepared, and 5 ml batches of themedium were placed in test tubes and sterilized. Brevibacteriumlactofermentum FERMP-1858 having requirements for methionine andisoleucine, which was obtained by a conventional mutant inducing method,for example a replica method, from parent strain Brevibacteriumlactofermentum ATCC 13869, was inoculated on the medium and cultured at31C for 16 hours.

Microbial cells harvested from the cultured broth were added into 5 mlof phosphate-buffer containing 250 ug/ml of nitrosoguanidine, and thebuffer solution with the cells was maintained at 31C for 30 minutesunder shaking. Thereafter, the microbial cells were harvested and washedtwice with phosphate buffer solution. V

The microbial cells treated as mentioned above were inoculated on anagar plate shown hereunder, and were cultured at 31C for 2 days.

Composition of a medium:

glucose 2 NH4 12 4 1 KH PO O. 1 MgSO,-7H O 0.04 Fe 2 m Mn 2 biotin 50#g/l thiamine'HCl urea (sterilized separately) 0.5 L-isoleucine 15 mg/dlDL-methionine 30 2-thiazolalanine 0.3

The 2-TA resistant strains appearing on the agar plate were isolated.Brevibacterium lactofermentum FERMP-1845 was isolated from saidresistant strains by examining their ability for producing L-valine.

Other mutant strains specified herebefore by FERMP-numbers were alsoobtained by the same method from the respective parent strains.

EXPERIMENT 2 amount shown in Table l, and cultivation was carried out at30C for 24 hours with shaking.

Basal medium:

glucose 2 urea (NH, ),,S0 0.

KH PO, 0.

biotin thiamine'HCl L-isoleucine DL-methionine Table l Conc. of 2-TARelative growth (y/ml) FERMP-l845 FERMP-l858 As is apparent from Tablel, the growth of Brevibacterium lactofermentum FERMP-1858 was inhibitedby addition of 2-TA, the growth of Brevibacterium lactofermentumFERMP-l845 in a medium containing 2,000 'y/ml of 2-TA was almost thesame as in a medium without 2-TA.

The mutant strain FERMP-l845 was found to grow even in a mediumcontaining 5,000 'y/ml of 2-TA.

The artificial mutants of Corynebacterium glutamicum were induced as inExperiment 1, and the growth of the mutants in a medium containing 2-TAwas studied by the same way shown in Experiment 2.

Corynebacterium glutamicum FERMP-l768 having a resistance to 2-TA wasinduced from Corynebacterium glutamicum ATCC 13032 by the methoddisclosed in Experiment 1. Cells of the strain FERMP- 1768 wereharvested after cultivation and were added to phosphate-buffercontaining 250 ug/ml of nitrosoguanidine. The solution was maintained at30C for minutes under shaking. After said microbial cells were washedtwice with the phosphate buffer solution, nutrient requiring mutantswere obtained by usual replica method. We could obtain many mutantstrains having a nutrient requirement together with resistance to 2-TA.Those mutant strains include Corynebacterium glutamicum FERMP-l967(leu'), Corynebacterium 6 glutamicum FERMP-l966 (isoleu) andCorynebacterium glutamicum FERMP-l968 (thr).

The growth inhibition of Corynebacterium glutamicum FERMP- l 768 by 2-TAwas examined as in Experiment 2. The relative growth of the parentstrain, Corynebacterium glutamicum ATCC 13032, in a medium containing1,000 y/ml of 2-TA became zero, and while the relative growth ofCorynebacterium glutamicum FERMP-l768 in a medium containing 2,000 'y/mlof 2-TA was more than and the strain could grow even in a mediumcontaining 5,000 'y/ml of 2-TA.

The culture medium used to produce L-valine in the present invention maybe entirely conventional. It should include an assimilable carbonsource, an assimilable nitrogen source, and the usual minor nutrients.Examples of the carbon source are carbohydrates such as glucose,maltose, fructose, starch, starch hydrolyzate, cellulose hydrolyzate ormolasses, organic acids such as acetic acid, propionic acid or succinicacid, alcohols such as ethanol or glycerol, and hydrocarbons such asn-paraffin. These substances may be used either singly or in mixtures oftwo or more. Useful nitrogen sources include ammonium sulfate, urea,ammonium nitrate, ammonium phosphate, ammonium chloride or gaseousammonia. Inorganic salts, such as phosphate, magnesium, calcium,ferrous, manganese and other minor metallic salts are generally present.For the nutrient requiring mutants, the nutrients required should bepresent. Amino acids, vitamins, soy-bean hydrolyzate, yeast extracts,peptone and caseine hydrolyzate are preferably present for goodbacterial growth.

The conditions for cultivation are quite conventional. The fermentationof the present invention is performed at a pH between 5 and 9, at atemperature of 20C to 40C under aerobic conditions for l to 4 days. ThepH of the culture medium can be adjusted by adding sterile calciumcarbonate, aqueous or gaseous ammonia, mineral acid or organic acidduring the fermentation.

The L-valine is recovered from the cultured broth by conventionalmethods. L-valine produced by the present method was identified by paperchromatography, electrophoresis, and response to microbioassay as wellas by comparision with an authentic sample.

The amount of L-valine produced in cultured broth was determined bymicrobioassay.

The following Examples are illustrative of the ,method of the presentinvention.

EXAMPLE 1 A culture medium whose composition is shown hereunder wasprepared, and 20 ml batches of the medium were each placed in a 500 mlshaking flask.

Composition of the medium:

glucose U2 4 KH P0, MgSOHH O F M biotin thiamine HCl soy bean proteinhydrolyzate CaCO (sterilized separately) (pH 7.0 adjusted by KOH) Theeight of strains shown in Table 2, which had previously been cultured onrespective bouillon slants at 30C for 24 hours, were introducedrespectively into each medium supplemented with a required amino acidTable 2 Amino acids required L-valine Note: All strains a have aresistance to Z-TA.

One liter each of the broth of the strain FERMP- 1964 and strainFERMP-l966 was centrifuged to remove bacterial cells, each supernatantwas passed through a column packed with a cation exchange resin, andafter washing with water, L-valine was eluted.

From, the From 9.3 g and 11.2 g of crystalline L- -valine wererecovered.

EXAMPLE 2 ml batches of the fermentation medium shown hereunder wereeach placed in a 500 ml shaking flask, and sterilized. The medium wasinoculated with Brevibacterium lactofermentum FERMP-l845 (Z-TA isoleuplus met) which had previously been cultured on a bouillon slant at Cfor 24 hours, and cultured at 30C for 72 hours with stirring andaerating. The cultured broth was found to contain 2.38 g/dl of L-valine.As a control, Brevibacterium lactofermentum FERMP- 1858 (isoleu' plusmet) having no resistance to 2-TA was cultured by the same way mentionedabove, and 0.9 g/dl of L-valine was found in the broth after 72 hourscultivation, the mutant strain FERM P-1845 having produced more thantwice as much valine.

Cut wroccpptsoc O- 6 L-isoleucinc 20 mg/dl DL-methionine 4O CaCO(sterilized separately) 5 7r What we claim is:

l. A method of producing L-valine by fermentation which comprises:

a. culturing a microorganism of the genera Brevibacterium orCorynebacterium on a nutrient medium under aerobic conditions,

1. said microorganism being capable of producing extracellular L-valinein said medium,

2. said medium containing sources of assimilable carbon and nitrogen andminor organic and inorganic nutrients necessary to the growth of saidmicroorganism, Y

3. said microorganism being cultured on said nutrient medium untilL-valine accumulates in said medium,

4. said microorganism being resistant to 2- thiazolalanine, and

b. recovering accumulated L-valine from said medium.

2. A method as set forth in claim 1, said microorganism requiring forgrowth in said medium at least one compound selected from the groupconsisting of leucine, isoleucine, threonine and methionine.

3. A method as set forth in claim 1, said microorganism being a strainof Brevibacterium lactofermentum, Corynebacterium glutamicum, orCorynebacterium acetoacidophilum.

4. A method as set forth in claim 1, wherein said microorganism isBrevibacterium lactofermentum FERMP-l 845, Brevibacterium lactofermentumFERMP- 1 945, Brevibacterium lactofermentum FERMP-1963, Brevibacteriumlactofermentum FERMP- l 964, Brevibacterium lactofermentum ent strain.

1. A METHOD OF PRODUCING L-VALINE BY FERMENTATION WHICH COMPRISES: A.CULTURING A MICROORGANISM OF THE GENERA BREVIBACTERIUM ORCORYNEBACTERIUM ON A NUTRIENT MEDIUM UNDER AEROVIC CONDITIONS,
 1. SAIDMICROORGANISM BEING CAPABLE OF PRODUCING EXTRACELLULAR L-VALINE IN SAIDMEDIUM,
 2. SAID MEDIUM CONTAINING SOURCES OF ASSIMILABLE CARBON ANDNITROGEN AND MINOR ORGANIC AND INORGANIC NUTRIENTS NECESSARY TO THEGROWTH OFF SAID MICROORGANISM,
 2. A method as set forth in claim 1, saidmicroorganism requiring for growth in said medium at least one compoundselected from the group consisting of leucine, isoleucine, threonine andmethionine.
 2. said medium containing sources of assimilable carbon andnitrogen and minor organic and inorganic nutrients necessary to thegrowth of said microorganism,
 3. said microorganism being cultured onsaid nutrient medium until L-valine accumulates in said medium,
 3. SAIDMICROORGANISM BEING CULTURED ON SAID NUTRIENT MEDIUM UNTIL L-VALINEACCUMULATES IN SAID MEDIUM,
 3. A method as set forth in claim 1, saidmicroorganism being a strain of Brevibacterium lactofermentum,Corynebacterium glutamicum, or Corynebacterium acetoacidophilum.
 4. SAIDMICROORGANISM BEING RESISTANT TO 2-THIAZOLALANINE, AND B. RECOVERINGACCUMULATED L-VALINE FROM SAID MEDIUM.
 4. said microorganism beingresistant to 2-thiazolalanine, and b. recovering accumulated L-valinefrom said medium.
 4. A method as set forth in claim 1, wherein saidmicroorganism is Brevibacterium lactofermentum FERMP-1845,Brevibacterium lactofermentum FERMP-1945, Brevibacterium lactofermentumFERMP-1963, Brevibacterium lactofermentum FERMP-1964, Brevibacteriumlactofermentum FERMP-1965, Corynebacterium glutamicum FERMP-1768,Corynebacterium glutamicum FERMP-1966, corynebacterium glutamicumFERMP-1967, Corynebacterium glutamicum FERMP-1968, or Corynebacteriumacetoacidophilum FERMP-1314.
 5. A method as set forth in claim 3,wherein said microorganism is an artificially induced mutant of a parentstrain lacking significant resistance to thiazolalanine, said mutantproducing extracellular valine in said medium in an amount which is morethan twice the amount of valine produced in said medium by said parentstrain.